Methods for Viral Particle Characterization Using Two-Dimensional Liquid Chromatography-Mass Spectrometry

ABSTRACT

Methods for identifying viral protein constituents and quantifying the relative abundance of such viral protein constituents in a sample of viral particles are disclosed. In embodiments, the methods include first-dimension chromatography to separate intact viral capsid components of the sample, online denaturation of the viral capsid components to produce intact viral proteins, second-dimension chromatography to separate the viral proteins, and mass spectrometry to determine the masses of the viral proteins and identify the viral protein constituents of the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of USProvisional Application Nos.: 63/220,651, filed Jul. 12, 2021;63/275,138, filed Nov. 3, 2021; 63/359,554, filed Jul. 8, 2022; and63/359,557, filed Jul. 8, 2022, each of which is incorporated herein byreference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to methods for characterization of qualityattributes of viral particles (e.g., AAV capsids) using atwo-dimensional liquid chromatography-mass spectrometry platform.

BACKGROUND

Adeno-associated virus (AAV), which is a non-enveloped, single-strandedDNA virus, has emerged as an attractive class of therapeutic agents todeliver genetic materials to host cells for gene therapy, due to itsability to transduce a wide range of species and tissue in vivo, lowrisk of immunotoxicity, and mild innate and adaptive immune responses.The complex nature of viral vectors such as AAV require specificanalytical methods to enable product testing and characterization.

Existing analytical techniques often do not provide sufficientresolution for quantifying homogeneity for the production ofclinical-grade viral vector preparations. Complete characterization ofthe constituent viral capsid proteins, such as the capsid proteins ofAAV vectors, including their sequences and post-translationalmodifications (PTMs), is desirable to ensure product quality andconsistency. Thus, methods are needed to determine the homogeneity ofviral particles and identify various species of viral proteins withinthe viral particles.

BRIEF SUMMARY OF THE INVENTION

The present disclosure is directed to an online two-dimensional liquidchromatography-mass spectrometry (2DLC-MS) platform for viral particle(e.g., AAV) characterization, which can simultaneously performcharacterization of the empty and full ratio and viral proteins bychromatographic separation of viral particles and viral proteins coupledwith mass spectrometry. In exemplary embodiments, characterization ofthe empty and full ratio and viral proteins is performed byanion-exchange chromatography (AEX) and reverse-phase liquidchromatography (RPLC) coupled with mass spectrometry (MS), respectively.

In one aspect, the present disclosure provides a method for identifyingviral protein constituents of a sample of viral particles, comprising:(a) subjecting the sample of viral particles to first-dimensionchromatography to separate intact viral capsid components of the sample;(b) subjecting at least a portion of the intact viral capsid componentsto online denaturation to yield individual intact viral proteins; (c)subjecting the intact viral proteins to second-dimension chromatographyto separate the intact viral proteins; and (d) determining the masses ofthe separated intact viral proteins to identify the viral proteinconstituents of the sample of viral particles.

In some embodiments, the method further comprises selecting a portion ofthe separated intact viral capsid components, wherein subjecting atleast a portion of the intact viral capsid components to onlinedenaturation to yield individual viral proteins comprising subjected theselected portion of the separated intact viral capsid components toonline denaturation.

In some embodiments, the sample of viral particles comprisesadeno-associated virus (AAV) particles. In some cases, the AAV particlesare of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV-DJ, AAV-DJ/8, AAV-Rh10, AAV-retro, AAV-PHP.B, AAV8-PHP.eB, orAAV-PHP.S. In some cases, the AAV particles are of serotype AAV1. Insome cases, the AAV particles are of serotype AAV5. In some cases, theAAV particles are of serotype AAV8.

In some embodiments, the intact viral capsid components comprise emptyviral capsids and full viral capsids.

In some embodiments, the first-dimension chromatography comprisesion-exchange chromatography. In some cases, the ion-exchangechromatography is anion-exchange chromatography.

In some embodiments, the second-dimension chromatography comprisesreverse-phase chromatography. In some embodiments, the second-dimensionchromatography comprises hydrophilic interaction liquid chromatography.

In some embodiments, determining the masses of the separated intactviral proteins comprises subjecting the separated intact viral proteinsto electrospray ionization mass spectrometry.

In some embodiments, the viral protein constituents comprise VP1, VP2and/or VP3 of an AAV particle. In some cases, the viral proteinconstituents comprise post-translational variants of VP1, VP2 and/orVP3. In some cases, the post-translational variants of VP1, VP2 and/orVP3 comprise acetylated, phosphorylated and/or oxidized variants of VP1,VP2 and/or VP3. In some cases, the post-translational variants of VP1,VP2 and/or VP3 comprise fragments of VP1, VP2 and/or VP3 produced fromcleavage of an aspartic acid-proline bond and/or cleavage of an asparticacid-glycine bond.

In some embodiments, the method further comprises detecting the intactviral capsid components separated by the first dimension chromatography,and identifying a ratio of empty viral capsids to full andpartially-full viral capsids.

In some embodiments, the method further comprises detecting the intactviral proteins separated by the second-dimension chromatography, andquantifying the relative abundance of the viral protein constituents ofthe sample of viral particles.

In some cases, the intact viral capsid components and/or the intactviral proteins are detected using an ultraviolet or fluorescencedetector.

In one aspect, the present disclosure provides a method for identifyingviral protein constituents of a sample of adeno-associated virus (AAV)particles, comprising: (a) subjecting the sample of AAV particles toanion-exchange chromatography to separate intact viral capsid componentsin the sample, wherein the intact viral capsid components compriseintact empty viral capsids and intact full viral capsids comprising aheterologous nucleic acid molecule; (b) selecting a portion of theintact viral capsid components for online desalting and denaturation;(c) subjecting the selected portion of the intact viral capsidcomponents to online desalting and denaturation to yield individualintact viral proteins, wherein the intact individual viral proteinscomprise VP1, VP2, VP3 and at least one variant of VP1, VP2 or VP3; (d)subjecting the intact viral proteins to reverse-phase liquidchromatography or hydrophilic interaction liquid chromatography toseparate the intact viral proteins; and (e) determining the masses ofthe separated intact viral proteins to identify the viral proteinconstituents of the sample of AAV particles.

In some embodiments, the intact viral proteins are subjected toreverse-phase liquid chromatography. In some embodiments, the intactviral proteins are subjected to hydrophilic interaction liquidchromatography.

In some embodiments, the method further comprises detecting the intactviral capsid components separated by the anion-exchange chromatography,and identifying a ratio of empty viral capsids to full andpartially-full viral capsids.

In some embodiments, the method further comprises detecting the intactviral proteins separated by the reverse-phase liquid chromatography orhydrophilic interaction liquid chromatography, and quantifying therelative abundance of the viral protein constituents of the sample ofAAV particles.

In some embodiments, the AAV particles are of serotype AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV-DJ/8, AAV-Rh10,AAV-retro, AAV-PHP.B, AAV8-PHP.eB, or AAV-PHP.S. In some cases, the AAVparticles are of serotype AAV1. In some cases, the AAV particles are ofserotype AAV5. In some cases, the AAV particles are of serotype AAV8.

In some embodiments, the at least one variant of VP1, VP2 or VP3comprises a post-translational variant of VP1, VP2 or VP3. In somecases, the post-translational variant of VP1, VP2 or VP3 comprises anacetylated variant of VP1, VP2 or VP3. In some cases, thepost-translational variant of VP1, VP2 or VP3 comprises a phosphorylatedvariant of VP1, VP2 or VP3. In some cases, the post-translationalvariant of VP1, VP2 or VP3 comprises an oxidized variant of VP1, VP2 orVP3. In some cases, the post-translational variant of VP1, VP2 or VP3comprises a fragment of VP1, VP2 or VP3 produced from cleavage of anaspartic acid-proline bond. In some cases, the post-translationalvariant of VP1, VP2 or VP3 comprises a fragment of VP1, VP2 or VP3produced from cleavage of an aspartic acid-glycine bond.

In some embodiments, the intact viral capsid components and/or theintact viral proteins are detected using an ultraviolet or fluorescencedetector.

In some embodiments, determining the masses of the separated intactviral proteins comprises subjecting the separated intact viral proteinsto electrospray ionization mass spectrometry.

In some embodiments, the intact viral capsid components of the samplesubjected to anion-exchange chromatography are separated using a firstmobile phase comprising from 15 mM to 25 mM bis-tris-propane (BTP), from250 mM to 1 M tetramethylammonium chloride (TMAC), and from 1 mM to 3 mMmagnesium chloride at a pH of from 8 to 9. In some cases, the firstmobile phase comprises 20 mM ±2 mM BTP, 500 mM ±50 mM TMAC, and 2 mM±0.2 mM MgCl₂ at a pH of 8.5±0.1. In some embodiments, the intact viralcapsid components of the sample subjected to anion-exchangechromatography are separated using the first mobile phase and a secondmobile phase comprising from 15 mM to 25 mM bis-tris-propane (BTP), andfrom 1 mM to 3 mM magnesium chloride at a pH of from 8 to 9. In somecases, the second mobile phase comprises 20 mM ±2 mM BTP, and 2 mM ±0.2mM MgCl₂ at a pH of 8.5±0.1. In some embodiments, the intact viralcapsid components of the sample subjected to anion-exchangechromatography are separated using the first mobile phase, the secondmobile phase, and a third mobile phase comprising from 1.5 M to 2.5 Msodium chloride. In some cases, the third mobile phase comprises 2 M±0.1 M sodium chloride. In some embodiments, the separation of theintact viral capsid components is performed with a mobile phasegradient. In some cases, the mobile phase gradient comprises, insequence: 10% first mobile phase and 90% second mobile phase for 1minute; increasing the first mobile phase from 10% to 42%, anddecreasing the second mobile phase from 90% to 58%, over a period of 20minutes; 100% third mobile phase for 5 minutes; and 10% first mobilephase and 90% second mobile phase for 10 minutes.

In some embodiments, the method further comprises identifying an amountof intact empty viral capsids and an amount of full viral capsids in thesample, and determining a relative abundance of the intact empty viralcapsids and intact full viral capsids in the sample.

In various embodiments, any of the features or components of embodimentsdiscussed above or herein may be combined, and such combinations areencompassed within the scope of the present disclosure. Any specificvalue discussed above or herein may be combined with another relatedvalue discussed above or herein to recite a range with the valuesrepresenting the upper and lower ends of the range, and such ranges areencompassed within the scope of the present disclosure.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 10 illustrate an AAV capsid comprising a heterologousnucleic acid molecule (e.g., a therapeutic gene or gene of interest(GOI)) (FIG. 1A); empty, partially-full and full capsids (FIG. 1B); anda AAV capsid composed of 60 copies of three viral proteins (VP1, VP2 andVP3) that give rise to a wide range of theoretical capsidstoichiometries (FIG. 10 ).

FIGS. 2A and 2B illustrate an exemplary two-dimensional liquidchromatography-mass spectrometry system (2DLC-MS) in accordance with anembodiment of the present disclosure, in which viral capsids areseparated in a first dimension and viral proteins are separated in asecond dimension for mass spectral analysis.

FIG. 2C illustrates a valve setup for a 2DLC-MS system in accordancewith an embodiment of the present disclosure. Part (a) illustrates afirst position for the valve setup in which a second liquidchromatography flow is used to maintain the RPLC column temperature, andone fraction from the trapping loop enters the trap column for desaltingand denaturation. Part (a) further illustrates a second position for thevalve setup in which viral proteins from the denatured viral capsid(e.g., AAV) is migrated from the trap column to the analytical column(e.g., RPLC) for separation followed by mass spectral analysis. Parts(b) (c) (d) illustrate the separation of viral proteins with (part (c))or without (part (b)) a trap column, and with different flow rates (0.2mL/min in part (c); and 0.1 mL/min in part(d)). As shown, greaterseparation of the viral proteins is achieved with use of the trapcolumn, and there is no significant change in the peaks with a change inflow rate. Part (e) shows that no salt adduct is observed in connectionwith the three AAV viral proteins (from deconvoluted spectra) using theexemplified valve setup.

FIG. 2D shows a pair of chromatograms demonstrating that onlinedenaturation (bottom chromatogram) provides for effective dissociationof the AAV viral proteins without the need for denaturation prior tosample injection.

FIG. 2E shows raw and deconvoluted spectra of the peak representing highmolecular weight species obtained from the 2DLC-MS system (forAAV8-GOI1), and confirming the identities of the high molecular weightspecies as the VP3 dimer and the VP2+VP3 heterodimer. Use of the trapcolumn for online denaturation eliminated the high molecular weightspecies (compare FIG. 2C, parts (b) and (c), which show the presence ofthe high molecular weight species without the trap column, and itsabsence with the trap column, respectively).

FIGS. 3A and 3B are representative of chromatograms obtained from thefirst-dimension chromatography (e.g., AEX), and show separation of viralcapsids (empty or containing a GOI) using either tetramethylammoniumchloride or tetraethylammonium chloride (FIG. 3A) and from various AAVserotypes (FIG. 3B), respectively.

FIGS. 3C and 3D are representative of chromatograms and a mass spectrumobtained from the second-dimension chromatography (e.g., RPLC) and massspectrometry, and show that viral proteins in AAV samples with orwithout a GOI can be effectively separated in both AAV8 and AAV1samples, and that the GOI did not interfere with the separation (FIG.3C), and that separation of viral proteins coupled with massspectrometry can be used to identify the viral proteins (FIG. 3D).

FIG. 4A illustrates a process in accordance with an embodiment of thepresent disclosure, showing (i) a chromatogram in which empty capsids(AAV8-Em) have been separated from capsids containing a gene of interest(AAV8-GOI), (ii) selection of a portion of the separated capsids fordenaturation (“heart cutting”), (iii) a chromatogram in which viralproteins (VP1, VP2, VP3 and others) have been separated from oneanother, and (iv) a mass spectrum corresponding to the separated viralproteins.

FIG. 4B illustrates multiple “heart cutting” of peaks followingfirst-dimension chromatography (e.g., AEX), followed by second-dimensionchromatography (e.g., RPLC) and mass spectrometry to identify andcharacterize the viral protein constituents of the peaks in an AAV8-GOIsample.

FIG. 5 illustrates the effectiveness of the first-dimensionchromatography in separating empty viral capsids from capsid containinga heterologous nucleic acid molecule (e.g., gene of interest or GOI).The chromatographic separation yields a ratio of viral capsids in which(i) the empty capsids, and (ii) the partially-full and full capsids areconsistent with data produced from analytical ultracentrifugation (AUC)techniques.

FIG. 6 illustrates the chromatographic separation of viral proteins viasecond-dimension chromatography and the relative quantity of each ofVP1, VP2 and VP3 (of AAV).

FIGS. 7A and 7B illustrate the chromatographic separation of viralproteins (VP1, VP2 and VP3 of AAV) and post-translational variants ofthe viral proteins via second-dimension chromatography. FIG. 7A showslabels for the abundant species, while FIG. 7B shows labels for thelow-abundant species.

FIG. 7C shows the identities of the species labeled in FIGS. 7A and 7Bfor an AAV8 sample of viral particles, along with the observed mass andtheoretical mass of each. “Ac” refers to acetylated, “P” refers tophosphorylated, “Clip (DP)” refers to a fragment produced by cleavage ofan aspartic acid-proline bond, “Ox” refers to an oxidized, and “Clip(DG)” refers to a fragment produced by cleavage of an asparticacid-glycine bond.

FIG. 7D shows the identities of species for an AAV1 sample of viralparticles in the same manner as FIG. 7C.

FIGS. 8A and 8B show mass spectra corresponding to the viral proteinconstituents of AAV8 or AAV1 capsids that are either empty or contain agene of interest (GOI).

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms “include,” “includes,” and “including,” aremeant to be non-limiting and are understood to mean “comprise,”“comprises,” and “comprising,” respectively.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

SELECTED ABBREVIATIONS

-   -   2DLC-MS— Two-Dimensional Liquid Chromatography-Mass Spectrometry    -   LC-MS— Liquid Chromatography-Mass Spectrometry    -   MS: Mass Spectrometry or Mass Spectrometer    -   ESI: Electrospray Ionization    -   rAAV: Recombinant AAV Particle or Capsid    -   AAV: Adeno-Associated Virus    -   LC: Liquid Chromatography    -   RPLC: Reverse Phase Liquid Chromatography    -   HILIC— Hydrophilic Interaction Liquid Chromatography    -   AEX— Anion Exchange Chromatography    -   IEX— Ion Exchange Chromatography    -   GOI— gene of interest    -   VP1— Viral Protein 1 subunit of AAV    -   VP2— Viral Protein 2 subunit of AAV    -   VP3— Viral Protein 3 subunit of AAV    -   UV— Ultraviolet    -   FLR— Fluorescence

Definitions

“Intact viral capsid components” refer to viral capsids (e.g., emptyviral capsids, partially-full viral capsids, and/or full viral capsids)that are intact (i.e., have not been denatured or otherwise broken downor disintegrated into their component parts (e.g., different viralproteins) and retain the structural characteristics of a viral capsid(e.g., the icosahedral conformation of an AAV capsid).

The terms “empty viral capsids” or “empty capsids” refer to capsids notcontaining a heterologous nucleic acid molecule (e.g., a therapeuticgene), as illustrated in FIG. 1B.

The terms “partially-full viral capsids” or “partially full capsids”refer to capsids containing only a portion of a heterologous nucleicacid molecule (e.g., a therapeutic gene), as illustrated in FIG. 1B.

The terms “full viral capsids” or “full capsids” refer to capsidscontaining a complete heterologous nucleic acid molecule (e.g., atherapeutic gene or gene of interest), as illustrated in FIG. 1B.

The term “sample,” as used herein, refers to a mixture of viralparticles (e.g., AAV particles) that comprises at least one viral capsidcomponent (i.e., empty capsids, partially-full capsids, and/or fullcapsids), that is subjected to manipulation in accordance with themethods of the invention, including, for example, separating andanalyzing.

The terms “analysis” or “analyzing,” are used interchangeably and referto any of the various methods of separating, detecting, isolating,purifying and/or characterizing viral particles or viral proteins ofinterest (e.g., AAV proteins). Examples include, but are not limited to,mass spectrometry, e.g., ESI-MS, liquid chromatography (e.g., AEX, RPLCor HILIC), and combinations thereof.

“Contacting,” as used herein, includes bringing together at least twosubstances in solution or solid phase, for example contacting astationary phase of a chromatography material with a sample, such as asample comprising viral particles or viral proteins.

“Intact mass analysis” as used herein includes experiments wherein aviral protein is characterized as an intact protein. Intact massanalysis can reduce sample preparation to a minimum.

As used herein, the term “liquid chromatography” refers to a process inwhich a chemical mixture carried by a liquid can be separated intocomponents as a result of differential distribution of the chemicalentities as they flow around or over a stationary liquid or solid phase.Non-limiting examples of liquid chromatography include reverse phaseliquid chromatography, ion-exchange chromatography, size exclusionchromatography, affinity chromatography, and hydrophobic interactionchromatography.

As used herein, the term “mass spectrometer” refers to a device capableof detecting specific molecular species and accurately measuring theirmasses. The term can be meant to include any molecular detector intowhich a viral protein (e.g., AAV protein) may be eluted for detectionand/or characterization. A mass spectrometer consists of three majorparts: the ion source, the mass analyzer, and the detector. The role ofthe ion source is to create gas phase ions. Analyte atoms, molecules, orclusters can be transferred into gas phase and ionized eitherconcurrently (as in electrospray ionization). The choice of ion sourcedepends on the application. As used herein, the term “electrosprayionization” or “ESI” refers to the process of spray ionization in whicheither cations or anions in solution are transferred to the gas phasevia formation and desolvation at atmospheric pressure of a stream ofhighly charged droplets that result from applying a potential differencebetween the tip of the electrospray emitter needle containing thesolution and a counter electrode. There are three major steps in theproduction of gas-phase ions from electrolyte ions in solution. Theseare: (a) production of charged droplets at the ES infusion tip; (b)shrinkage of charged droplets by solvent evaporation and repeateddroplet disintegrations leading to small highly charged droplets capableof producing gas-phase ions; and (c) the mechanism by which gas-phaseions are produced from very small and highly charged droplets. Stages(a)-(c) generally occur in the atmospheric pressure region of theapparatus.

As used herein, the term “electrospray ionization source” refers to anelectrospray ionization system that can be compatible with a massspectrometer used for mass analysis of viral particles.

Native MS is a particular approach based on electrospray ionization inwhich the biological analytes are sprayed from a nondenaturing solvent.It is defined as the process whereby biomolecules, such as largebiomolecules, and complexes thereof can be transferred from athree-dimensional, functional existence in a condensed liquid phase tothe gas phase via the process of electrospray ionization massspectrometry (ESI-MS).

The term “nanoelectrospray” or “nanospray” as used herein refers toelectrospray ionization at a very low solvent flow rate, typicallyhundreds of nanoliters per minute of sample solution or lower, oftenwithout the use of an external solvent delivery.

As used herein, “mass analyzer” refers to a device that can separatespecies, that is, atoms, molecules, or clusters, according to theirmass. Non-limiting examples of mass analyzers that could be employed forfast protein sequencing are time-of-flight (TOF), magnetic/electricsector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbitrap,Fourier transform ion cyclotron resonance (FTICR), and also thetechnique of accelerator mass spectrometry (AMS).

As used herein, “mass-to-charge ratio” or “m/z” is used to denote thedimensionless quantity formed by dividing the mass of an ion in unifiedatomic mass units by its charge number (regardless of sign).

As used herein, the term “quadrupole—Orbitrap hybrid mass spectrometer”refers to a hybrid system made by coupling a quadrupole massspectrometer to an orbitrap mass analyzer. A tandem in-time experimentusing the quadrupole—Orbitrap hybrid mass spectrometer begins withejection of all ions except those within a selected, narrow m/z rangefrom the quadrupole mass spectrometer. The selected ions can be insertedinto orbitrap and fragmented most often by low-energy CID. Fragmentswithin the m/z acceptance range of the trap should remain in the trap,and an MS-MS spectrum can be obtained.

“Adeno-associated virus” or “AAV” is a non-pathogenic parvovirus, withsingle-stranded DNA, a genome of approximately 4.7 kb, not enveloped andhas icosahedric conformation. AAV was first discovered in 1965 as acontaminant of adenovirus preparations. AAV belongs to the Dependovirusgenus and Parvoviridae family, requiring helper functions from eitherherpes virus or adenovirus for replication. In the absence of helpervirus, AAV can set up latency by integrating into human chromosome 19 atthe 19q13.4 location. The AAV genome consists of two open reading frames(ORF), one for each of two AAV genes, Rep and Cap. The AAV DNA ends havea 145-bp inverted terminal repeat (ITR), and the 125 terminal bases arepalindromic, leading to a characteristic T-shaped hairpin structure.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. Thus, this term includes, but is not limited to,single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. The backbone of the nucleic acid cancomprise sugars and phosphate groups (as may typically be found in RNAor DNA), or modified or substituted sugar or phosphate groups.

A “recombinant viral particle” refers to a viral particle including oneor more heterologous sequences (e.g., a nucleic acid sequence not viralorigin) that may be flanked by at least one viral nucleotide sequence.

A “recombinant AAV particle” refers to a adeno-associated viral particleincluding one or more heterologous sequences (e.g., nucleic acidsequence not of AAV origin) that may be flanked by at least one, forexample, two, AAV inverted terminal repeat sequences (ITRs). Such rAAVparticles can be replicated and packaged when present in a host cellthat has been infected with a suitable helper virus (or that isexpressing suitable helper functions) and that is expressing AAV rep andcap gene products (i.e., AAV Rep and Cap proteins).

A “viral particle” refers to a viral particle composed of at least oneviral capsid protein and an encapsulated viral genome.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is compared or into which itis introduced or incorporated. For example, a nucleic acid introduced bygenetic engineering techniques into a different cell type is aheterologous nucleic acid (and, when expressed, can encode aheterologous polypeptide). Similarly, a cellular sequence (e.g., a geneor portion thereof) that is incorporated into a viral particle is aheterologous nucleotide sequence with respect to the viral particle.

An “inverted terminal repeat” or “ITR” sequence is relatively shortsequences found at the termini of viral genomes which are in oppositeorientation. An “AAV inverted terminal repeat (ITR)” sequence, is anapproximately 145-nucleotide sequence that is present at both termini ofa single-stranded AAV genome.

The term “corresponding” is a relative term indicating similarity inposition, purpose or structure. A mass spectral signal due to aparticular peptide or protein is also referred to as a signalcorresponding to the peptide or protein. In certain embodiments, aparticular peptide sequence or set of amino acids, such as a protein,can be assigned to a corresponding peptide mass.

The term “isolated,” as used herein, refers to a biological component(such as a nucleic acid, peptide, protein, lipid, viral particle ormetabolite) that has been substantially separated, produced apart from,or purified away from other biological components in the cell of theorganism in which the component naturally occurs or is transgenicallyexpressed.

The terms “peptide,” “protein” and “polypeptide” refer, interchangeably,to a polymer of amino acids and/or amino acid analogs that are joined bypeptide bonds or peptide bond mimetics. The twenty naturally-occurringamino acids and their single-letter and three-letter designations are asfollows: Alanine A Ala; Cysteine C Cys; Aspartic Acid D Asp; Glutamicacid E Glu; Phenylalanine F Phe; Glycine G Gly; Histidine H His;Isoleucine I He; Lysine K Lys; Leucine L Leu; Methionine M Met;Asparagine N Asn; Proline P Pro; Glutamine Q Gln; Arginine R Arg; SerineS Ser; Threonine T Thr; Valine V Val; Tryptophan w Trp; and Tyrosine YTyr.

References to a mass of an amino acid means the monoisotopic mass oraverage mass of an amino acid at a given isotopic abundance, such as anatural abundance. In some examples, the mass of an amino acid can beskewed, for example, by labeling an amino acid with an isotope. Somedegree of variability around the average mass of an amino acid isexpected for individual single amino acids based on the exact isotopiccomposition of the amino acid. The masses, including monoisotopic andaverage masses for amino acids are easily obtainable by one of ordinaryskill the art.

Similarly, references to a mass of a peptide or protein means themonoisotopic mass or average mass of a peptide or protein at a givenisotopic abundance, such as a natural abundance. In some examples, themass of a peptide can be skewed, for example, by labeling one or moreamino acids in the peptide or protein with an isotope. Some degree ofvariability around the average mass of a peptide is expected forindividual single peptides based on the exact isotopic composition ofthe peptide. The mass of a particular peptide can be determined by oneof ordinary skill the art.

A “vector,” as used herein, refers to a recombinant plasmid or virusthat comprises a nucleic acid to be delivered into a host cell, eitherin vitro or in vivo.

A “recombinant viral vector” refers to a recombinant polynucleotidevector including one or more heterologous sequences (i.e., nucleic acidsequence not of viral origin).

The term “hydrophilic interaction chromatography” or HILIC is intendedto include a process employing a hydrophilic stationary phase and ahydrophobic organic mobile phase in which hydrophilic compounds areretained longer than hydrophobic compounds. In certain embodiments, theprocess utilizes a water-miscible solvent mobile phase.

The term “reverse-phase liquid chromatography” or RPLC is intended toinclude a process that separates analytes based on nonpolar interactionsbetween analytes and a stationary phase (e.g., substrate). The nonpolaranalyte associates with and is retained by the nonpolar stationaryphase. Adsorption strengths increase with analyte nonpolarity, and theinteraction between the nonpolar analyte and the nonpolar stationaryphase (relative to the mobile phase) increases the elution time. Use ofmore nonpolar solvents in the mobile phase will decrease the retentiontime of the analytes, while more polar solvents tend to increaseretention times.

The term “anion-exchange chromatography” or AEX is intended to include aprocess that separates substances based on their charges using anion-exchange resin containing positively charged groups, such asdiethyl-aminoethyl groups. In solution, the resin is coated withpositively charged counter-ions.

General Description

The present disclosure provides two-dimensional liquid chromatographyand native mass spectrometry (MS) methods that provide sensitive andrapid identification and quantitative characterization of the viralprotein constituents of a sample of viral particles (e.g., AAVparticles). Complete characterization of the viral protein constituentsof viral particle compositions, such as the viral protein constituentsof viral capsid components of a sample of AAV particles, is necessary toensure product quality and consistency to maintain safety and efficacyof the compositions.

Recombinant viral vector compositions (e.g., AAV vector compositions)can contain varying levels of viral proteins and post-translationalmodifications of such viral proteins arising from various production,purification and storage conditions. The present methods provideanalytical techniques to identify and quantitate ratios of viral capsidcomponents in a sample of viral particles, and to identify andquantitate viral protein constituents of the viral particles, includinglow-abundant viral protein constituents comprising acetylated,phosphorylated, oxidized, and fragmented variants of viral proteins.

Methods for Identifying and Quantifying Viral Protein Constituents

Aspects of the disclosure are directed to methods for identifying andquantifying viral protein constituents in a sample of viral particles(e.g., recombinant AAV particles) in a two dimensional liquidchromatography-mass spectrometry (2DLC-MS) system.

In some cases, the method comprises: (a) subjecting the sample of viralparticles to first-dimension chromatography to separate intact viralcapsid components of the sample; (b) subjecting at least a portion ofthe intact viral capsid components to online denaturation to yieldindividual intact viral proteins; (c) subjecting the intact viralproteins to second-dimension chromatography to separate the intact viralproteins; and (d) determining the masses of the separated intact viralproteins to identify the viral protein constituents of the sample ofviral particles.

In some cases, the method comprises: (a) subjecting the sample of AAVparticles to anion-exchange chromatography to separate intact viralcapsid components in the sample, wherein the intact viral capsidcomponents comprise intact empty viral capsids and intact full viralcapsids comprising a heterologous nucleic acid molecule; (b) selecting aportion of the intact viral capsid components for online desalting anddenaturation; (c) subjecting the selected portion of the intact viralcapsid components to online desalting and denaturation to yieldindividual intact viral proteins, wherein the intact individual viralproteins comprise VP1, VP2, VP3 and at least one variant of VP1, VP2 orVP3; (d) subjecting the intact viral proteins to reverse-phase liquidchromatography or hydrophilic interaction liquid chromatography toseparate the intact viral proteins; and (e) determining the masses ofthe separated intact viral proteins to identify the viral proteinconstituents of the sample of AAV particles.

In various embodiments of the methods, the viral protein constituentscomprise viral proteins and post-translational variants of the viralproteins. For example, in compositions of AAV particles, the viralprotein constituents comprise viral proteins VP1, VP2 and VP3, andpost-translational variants of VP1, VP2 and/or VP3, including, in somecases, acetylated, phosphorylated and/or oxidized variants of VP1, VP2and/or VP3, and/or fragments of VP1, VP2 and/or VP3 produced fromcleavage of a peptide bond (e.g., cleavage of an aspartic acid-prolinebond and/or cleavage of an aspartic acid-glycine bond).

In the methods disclosed herein, the 2DLC-MS system is exemplified bythe schematic illustrated in FIG. 2A and 2B. In the example shown inFIG. 2B, the 2DLC-MS system 100 includes a first-dimension liquidchromatography column 102 (e.g., an AEX column) into which the sample ofviral particles 101 (e.g., AAV particles) is introduced to separate theviral capsid components of the sample from one another, a detector 104(e.g., a FLR detector) for detecting the eluate from the first-dimensioncolumn 102, peak-picking or heart-cutting software 106 to enableselection of a portion of the eluted and separated viral capsidcomponents of the sample, a trapping loop 108 for online desalting anddenaturation, and for temporarily storing the selected viral capsidcomponents, which are to be transferred to the second-dimensionchromatography column, a second-dimension chromatography column 110(e.g., a RPLC column) into which the selected viral capsid componentsare transferred to yield intact viral proteins from the viral capsid(e.g., via a starting mobile phase) before a gradient is applied toseparate the intact viral proteins from one another, a detector 112(e.g., a FLR detector) for detecting the eluate from thesecond-dimension column 110, and a mass spectrometer 114 (e.g., anESI-MS) to determine the mass of the separated viral proteins andthereby identify the viral protein constituents of the sample of viralparticles 101 in a mass spectrum 116. One advantage of the 2DLC-MSsystem discussed herein in the capability of incorporatingMS-incompatible salts for high-resolution separation in the firstdimension, before using MS-compatible reagents in the second dimensionfor MS characterization. This advantage can be achieved, for example,using the valve setup exemplified in FIG. 2C. As shown in FIG. 2D,online denaturation allows for efficient separation of the viralproteins.

In various embodiments of the methods discussed herein, separation ofthe viral capsid components (e.g., empty and full capsids) in thefirst-dimension chromatography can be used to determine the relativequantities of the capsid components within the sample of viralparticles. In the context of AEX chromatography, for example, emptyviral capsids (i.e., those not containing a heterologous nucleic acidmolecule) will elute before partially-full or full viral capsids becausethe negatively charged nucleic acids (e.g., DNA) encapsulated within thepartially-full and full viral capsids result in lower isoelectric point(p1) values and higher affinity for the AEX resins, which are positivelycharged. An example of this separation is illustrated in FIG. 3B, whichshows the AAV1, AAV5 and AAV8 empty capsids eluting prior to the capsidscontaining a gene of interest (GOI). Such separation can be achievedusing, for example, tetramethylammonium chloride or tetraethylammoniumchloride, as shown in FIG. 3A. As illustrated in FIG. 5 , detection ofthe eluted viral capsid components (e.g., via a fluorescence detector)can then be used to determine the ratio of empty capsids to capsidscontaining a GOI. These data are consistent with data generated from AUCmeasurements, which is generally regarded as a state-of-art techniquefor determining relative quantities of empty, partial, and full viralcapsid components (note that the partially-full and full viral capsidcomponents are combined in the AEX column of the table shown in FIG. 5).

In various embodiments, the methods of the present disclosure can beused to determine the identity and stoichiometry of various viralprotein constituents contained within the viral particles of a samplesubjected to the 2DLC-MS system. In embodiments, the separated viralcapsid components from the first-dimension chromatography are subjectedto denaturation to yield intact viral proteins that formerly comprisedthe viral capsid (e.g., VP1, VP2 and VP3 of an AAV capsid). The intactviral proteins are then subjected to second-dimension chromatography toseparate the viral proteins, which may include modified variants of theviral proteins (e.g., post-translational variants arising naturally, orfrom production, purification or storage conditions). Examples of thisseparation are illustrated in FIG. 3C, which shows that the presence ofa GOI does not impact the separation of viral proteins, and in FIG. 3D(top), which shows the viral proteins of an empty AAV8 capsid that havebeen separated on a RPLC column. The chromatogram in FIG. 3D shows thepeaks for the three natural viral proteins of an AAV capsid (VP1, VP2and VP3) as well as for a variant of VP3 produced from cleavage of apeptide bond (unspecified).

The separated viral proteins are then subjected to mass spectrometry toascertain the identities of the various viral proteins. An example massspectrum is illustrated in FIG. 3D (bottom), which shows theidentification of a VP2 viral protein and a phosphorylated VP2 viralprotein (from an AAV capsid). Further identification of the relativeratios of the viral proteins and identification of viral proteins andvariants is illustrated in FIGS. 6, 7A, 7B, 7C and 7D.

In embodiments of the methods discussed herein, it is also possible toselect a subset of the viral capsid components separated in thefirst-dimension chromatography for denaturation and separation/analysisin the second-dimension chromatography and mass spectrometry portions ofthe 2DLC-MS system. As illustrated in FIG. 4A, “heart-cutting” can beperformed to select a specified portion of eluate from thefirst-dimension chromatography for further processing in thesecond-dimension chromatography, and subsequent mass spectrometry. Thistechnique enables improved resolution and analysis of specificcomponents, such as low-abundant species of viral proteins that may bepresent in the sample under investigation. Multiple “heart cutting” canalso be performed to analyze various peaks from the first-dimensionchromatography, as shown in FIG. 4B.

The methods discussed herein include subjection a sample of viralproteins to reverse phase liquid chromatography (RPLC) or hydrophilicinteraction liquid chromatography (HILIC) to separate the proteincomponents of the viral capsid of the viral particles, such as viralparticles of interest where information about the capsid is desired. Inembodiments, a RPLC or a HILIC column is contacted with the intact viralproteins following first-dimension chromatography and denaturation. Incertain embodiments the method includes determining the masses ofprotein components of the viral capsid to identify the proteincomponents separated by the second-dimension chromatography (e.g., RPLCor HILIC), for example, using mass spectrometry techniques, such asthose described herein. In embodiments, the method includes calculatingthe relative abundance of the protein components of the viral capsidfrom the separation to determine the stoichiometry of protein componentsof a viral capsid of a viral particle, for example using ultraviolet(UV) detection or fluorescence (FLR) detection of the protein componentsof the viral capsid as they are eluted from the RPLC or HILIC column.For example, the area of a UV or FLR peak can be used to determine therelative abundance of the capsid proteins and used to calculate thestoichiometry of the capsid proteins in the viral capsid. In anotherexample, the peak height and/or peak UV or FLR intensity is used todetermine relative abundance. In some embodiments, the retention time ofthe different proteins on the second-dimension chromatography column(e.g., RPLC or HILIC) is determined as a function of the mobile phaseused and, in subsequent analysis this retention time can be used todetermine the proteins and relative abundance of the proteins from theviral particle without the need to determine the mass and/or identity ofthe proteins every time a determination of stoichiometry is made, e.g. astandard value or values can be developed. In some cases, the seconddimension chromatography column can be used for both denaturation andseparation of the viral protein components. In some cases, the methodsdiscussed herein can be used to determine the serotype of a viralparticle. For example, the masses of VP1, VP2 and VP3 of each AAVserotype are unique and can be used to identify or differentiate AAVcapsid serotypes. In addition, the separated capsid proteins can besubjected to downstream analysis, such as a determination of proteinsequence and post-translational modifications of the capsid proteins,for example with accurate mass measurement at the intact protein level.

In some embodiments of the methods discussed herein, the methods can beused to determine the heterogeneity of protein components in a capsid ofa viral particle. In embodiments, the method includes subjecting theviral particle to first-dimension chromatography to separate the viralcapsid components, and subjecting at least a portion of the viral capsidcomponents to second-dimension chromatography to separate the proteincomponents of the viral particle capsid. In embodiments, the methodincludes determining the masses of protein components of the viralcapsid. In some cases, the masses of the protein components of the viralcapsid are compared with theoretical masses of the viral capsid. Adeviation of one or more of the masses of protein components of theviral capsid indicates that one or more proteins of the capsid areheterogeneous. Conversely, no deviation would indicate that the proteinsof the capsid are homogeneous. In embodiments, heterogeneity is due toone or more of mixed serotypes, variant capsids, capsid amino acidsubstitutions, truncated capsids, or modified capsids. In someembodiments, the determination of the stoichiometry of proteincomponents of a viral capsid of a viral particle and the determinationof the heterogeneity of protein components in a capsid of a viralparticle are done on the same sample.

In certain embodiments, the viral particle is an adeno-associated virus(AAV) particle and the methods disclosed can be used to determine theidentity (and optionally stoichiometry) of protein components in acapsid of an AAV particle and/or heterogeneity of protein components ina capsid of an AAV particle. In embodiments, the protein components ofthe protein capsid comprise VP1, VP2 and VP3 of an AAV particle, as wellas one or more variants of VP1, VP2 or VP3. In embodiments, the AAVparticle is a recombinant AAV (rAAV) particle. In embodiments, the AAVparticle includes an AAV vector encoding a heterologous transgene. Insome embodiments, a determined or calculated mass of the presentdisclosure (e.g., the determined or calculated mass of VP1, VP2 and/orVP3, or variants thereof, of the AAV particle) may be compared with areference, for example, a theoretical mass of a VP1, VP2, and/or VP3, orvariants thereof, of one or more AAV serotypes. A reference may includea theoretical mass of a VP1, VP2, and/or VP3, or variants thereof, ofone or more of any of the AAV serotypes. For example, in someembodiments, the masses of VP1, VP2, and/or VP3, or variants thereof,are compared to theoretical masses of one or more of an AAV1 capsid, anAAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9capsid, an AAV 10 capsid, an AAV 11 capsid, an AAV 12 capsid, or avariant thereof. In some embodiments, a determined or calculated mass(e.g., the determined or calculated mass of VP1, VP2 and/or VP3 of theAAV particle) may be compared with a theoretical mass of a VP1, VP2,and/or VP3 of the corresponding AAV serotype.

Viral Particles

In certain aspects, the viral particle is an AAV particle and themethods disclosed can be used to determine the relative abundance ofviral capsid components in a sample of AAV particles, as well as theidentity and stoichiometry of viral protein constituents of a viralcapsid. The AAV particles may be recombinant AAV (rAAV) particles. TherAAV particle includes an AAV vector encoding a heterologous transgeneor heterologous nucleic acid molecule.

In certain aspects, the AAV particles include an AAV1 capsid, an AAV2capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid,an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, anAAV10 capsid, an AAV11 capsid, an AAV 12 capsid, or a variant thereof.In certain aspects, the AAV particles are of serotype AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV-DJ/8, AAV-Rh10,AAV-retro, AAV-PHP.B, AAV8-PHP.eB, or AAV-PHP.S. In some embodiments,the AAV particles are of serotype AAV1 or AAV8.

While AAV was the model viral particle for this disclosure, it iscontemplated that the disclosed methods can be applied to characterize avariety of viruses, for example, the viral families, subfamilies, andgenera. The methods of the present disclosure may find use, for example,in characterizing viral particles to monitor or detect relativeabundance of viral capsid components, and identities and stoichiometriesof viral protein components of the viral capsids, in a composition ofviral particles during production, purification or storage of suchcompositions.

In exemplary embodiments, the viral particle belongs to a viral familyselected from the group consisting of Adenoviridae, Parvoviridae,Retroviridae, Baculoviridae, and Herpesviridae.

In certain aspects, the viral particle belongs to a viral genus selectedfrom the group consisting of Atadenovirus, Aviadenovirus,lchtadenovirus, Mastadenovirus, Siadenovirus, Ambidensovirus,Brevidensovirus, Hepandensovirus, lteradensovirus, Penstyldensovirus,Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus,Dependoparvovirus, Erythroparvovirus, Protoparvovirus, Tetraparvovirus,Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus,Gammaretrovirus, Lentivirus, Spumavirus, Alphabaculovirus,Betabaculovirus, Deltabaculovirus, Gammabaculovirus, Iltovirus,Mardivirus, Simplexvirus, Varicellovirus, Cytomegalovirus,Muromegalovirus, Proboscivirus, Roseolovirus, Lymphocryptovirus,Macavirus, Percavirus, and Rhadinovirus.

In certain aspects, the Retroviridae is Moloney murine sarcoma virus(MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus(FLV), Spumavirus, Friend virus, Murine Stem Cell Virus (MSCV) RousSarcoma Virus (RSV), human T cell leukemia viruses, HumanImmunodeficiency Viruse (HIV), feline immunodeficiency virus (FIV),equine immunodeficiency virus (EIV), visna-maedi virus; caprinearthritis-encephalitis virus; equine infectious anemia virus; felineimmunodeficiency virus (FIV); bovine immune deficiency virus (BIV); orsimian immunodeficiency virus (SIV).

In some aspects, the viral particle (e.g., AAV particle) contains aheterologous nucleic acid molecule (e.g., a therapeutic gene or gene ofinterest). In some aspects, the heterologous nucleic acid molecule isoperably linked to a promoter. Exemplary promoters include, but are notlimited to, the cytomegalovirus (CMV) immediate early promoter, the RSVLTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, asimian virus 40 (SV40) promoter and a CK6 promoter, a transthyretinpromoter (TTR), a TK promoter, a tetracycline responsive promoter (TRE),an HBV promoter, an hAAT promoter, a LSP promoter, chimericliver-specific promoters (LSPs), the E2F promoter, the telomerase(hTERT) promoter; the cytomegalovirus enhancer/chickenbeta-actin/Rabbit.beta.-globin promoter and the elongation factor1-alpha promoter (EF1-alpha) promoter. In some aspects, the promotercomprises a human .beta.-glucuronidase promoter or a cytomegalovirusenhancer linked to a chicken .beta.-actin (CBA) promoter. The promotercan be a constitutive, inducible or repressible promoter. In someaspects, the invention provides a recombinant vector comprising anucleic acid encoding a heterologous transgene of the present disclosureoperably linked to a CBA promoter. In some cases, the native promoter,or fragment thereof, for the transgene will be used. The native promotercan be used when it is desired that expression of the transgene shouldmimic the native expression. The native promoter may be used whenexpression of the transgene must be regulated temporally ordeveloEmentally, or in a tissue-specific manner, or in response tospecific transcriptional stimuli. In a further aspect, other nativeexpression control elements, such as enhancer elements, polyadenylationsites or Kozak consensus sequences may also be used to mimic the nativeexpression.

Two Dimensional Liquid Chromatography-Mass Spectrometry (2DLC-MS) System

The methods disclosed herein include subjecting a viral particle totwo-dimensional liquid chromatography/mass spectrometry (2DLC-MS). As isknown in the art, LC/MS utilizes liquid chromatography for physicalseparation of ions and mass spectrometry for generation of mass spectraldata from the ions. Such mass spectral data may be used to determine,for example, molecular weight or structure, identification of particlesby mass, quantity, purity, and so forth. These data may representproperties of the detected ions such as signal strength (e.g.,abundance) over time (e.g., retention time), or relative abundance overmass-to-charge ratio. The exemplary 2DLC-MS system illustrated in FIG.2B can be used to determine relative abundance of viral capsidcomponents in a sample of viral particles, and to identify and quantifyviral protein constituents of the viral capsids (or a portion thereof).However, modifications to the illustrated exemplary system can also beemployed to determine relative abundance of intact viral capsidcomponents, and to identify and quantify viral protein constituents ofthe viral capsids.

Non-limiting examples of the first-dimension and second-dimension liquidchromatography columns 102 and 110 (see FIG. 2B) include reverse phaseliquid chromatography, ion-exchange chromatography, size exclusionchromatography, affinity chromatography, hydrophilic-interactionchromatography, and hydrophobic chromatography. Liquid chromatography,including HPLC, can be used to separate components of a sample of viralparticles into viral capsid components, and to separate viral proteincomponents of the viral capsids for further analysis. In someembodiments, the first-dimension chromatography comprises anion-exchangechromatography, and the second dimension chromatography comprisesreverse-phase liquid chromatography. In some embodiments, thefirst-dimension chromatography comprises anion-exchange chromatography,and the second dimension chromatography comprises hydrophilicinteraction liquid chromatography.

In various embodiments, the first-dimension chromatography comprisesanion-exchange chromatography employing a mobile phase A containing 20mM bis-tris propane in water, and a mobile phase B containing 20 mMbis-tris propane and 1 M tetraalkylammonium salt (e.g.,tetramethylammonium chloride or tetraethylammonium chloride). In somecases, the tetraalkylammonium salt is present at a concentration of fromabout 0.1 M to about 10 M. In various embodiments, thetetraalkylammonium salt is present at a concentration of about 0.5 M,about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1 M, about 1.1M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M,about 1.7 M, about 1.8 M, about 1.9 M, about 2 M, about 2.5 M, about 3M, about 3.5 M, about 4 M, about 4.5 M, about 5 M, about 6 M, about 7 M,about 8 M, about 9 M, or about 10 M. In some embodiments, mobile phaseA, mobile phase B, or both mobile phase A and mobile phase B compriseabout 1 M sodium chloride. In various embodiments, the sodium chlorideis present at a concentration of from about 0.1 M to about 10 M. Invarious embodiments, the sodium chloride is present at a concentrationof about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M,about 1 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2 M, about2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, about 5 M, about6 M, about 7 M, about 8 M, about 9 M, or about 10 M. In someembodiments, mobile phase A or mobile phase B, or both, contain a halidesalt of an alkali metal or an alkaline earth metal (e.g., chloride,bromide or iodide salts of sodium, potassium, lithium, calcium ormagnesium) at any of the concentration noted above. In some cases, thepH of the mobile phases is from about 7 to about 12. In some cases, thepH of the mobile phases is from about 8 to about 11. In some cases, thepH of the mobile phases is about 9, about 9.1, about 9.2, about 9.3,about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, orabout 10. In some embodiments, the flow rate is about 0.1 mL/min orabout 0.2 mL/min or about 0.3 mL/min.

In various embodiments, the second-dimension chromatography comprisesreverse-phase liquid chromatography or hydrophilic interaction liquidchromatography. In some cases, the second-dimension chromatographycomprises reverse-phase liquid chromatography employing a mobile phase Acontaining 0.1% to 0.5% difluoroacetic acid (DFA) in water, and a mobilephase B containing 0.1% to 0.5% DFA in acetonitrile (ACN). In variousembodiments, the DFA concentration is about 0.1%, about 0.15%, about0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, orabout 0.5%. In some embodiments, the flow rate is about 0.1 mL/min orabout 0.2 mL/min or about 0.3 mL/min.

In various embodiments, the eluate from the first-dimension and/orsecond-dimension chromatography is detected using UV of FLR detectors.In some cases, the FLR detectors utilized an excitation wavelength offrom about 260 nm to about 300 nm (e.g., about 280 nm) and an emissionwavelength of from about 310 to about 370 nm (e.g., about 330 nm orabout 350 nm).

In various embodiments, denaturation of the viral capsid components (orportion thereof) is performed with about 10% acetic acid. In someembodiments, denaturation is accomplished in the second-dimensionchromatography column by applying a starting mobile phase for a periodof time (e.g., about 10 min.) before applying a gradient to separate theintact viral proteins produced from the denaturation process. In someembodiments, the starting mobile phase comprises 80% mobile phase A and20% mobile phase B, wherein mobile phase A comprises 0.1% to 0.5%difluoroacetic acid (DFA) in water, and mobile phase B comprises 0.1% to0.5% DFA in acetonitrile.

In some embodiments, the mobile phase of the first-dimensionchromatography and/or the second-dimension chromatography is an aqueousmobile phase. In exemplary embodiments, the mobile phase used to elutethe viral proteins from the second-dimension chromatography is a mobilephase that is compatible with a mass spectrometer. In some exemplaryembodiments, the mobile phase used in the first or second-dimensionliquid chromatography column can include water, acetonitrile,difluoroacetic acid, or combinations thereof. The mobile phase mayinclude buffers with or without ion pairing agents, e.g., acetonitrileand water. Ion pairing agents include acetate, diifluoroacetic acid andsalts. Gradients of the buffers can be used, for example, if two buffersare used, the concentration or percentage of the first buffer candecrease while the concentration or percentage of the second bufferincreases over the course of the chromatography run. For example, thepercentage of the first buffer can decrease from about 100%, about 99%,about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about65%, about 60%, about 50%, about 45%, or about 40% to about 0%, about1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,about 35%, or about 40% over the course of the chromatography run. Asanother example, the percentage of the second buffer can increase fromabout 0%, about 1%, about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, or about 40% to about 100%, about 99%, about95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%,about 60%, about 50%, about 45%, or about 40% over the course of thesame run. In certain aspects, the proportion of mobile phase A in thechromatography increases over time. Optionally, the concentration orpercentage of the first and second buffer can return to their startingvalues at the end of the chromatography run. The percentages can changegradually as a linear gradient or in a non-linear (e.g., stepwise)fashion. For example, the gradient can be multiphasic, for example,biphasic, triphasic, etc.

In some exemplary embodiments, the mobile phase can have a flow ratethrough the liquid chromatography column of about 0.1 μL/min to about100 mL/min, or about 0.05 mL/min to about 5 mL/min. In some cases, theflow rate is about 0.05 mL/min, about 0.06 mL/min, about 0.07 mL/min,about 0.08 mL/min, about 0.09 mL/min, about 0.1 mL/min, about 0.11mL/min, about 0.12 mL/min, about 0.13 mL/min, about 0.14 mL/min, about0.15 mL/min, about 0.16 mL/min, about 0.17 mL/min, about 0.18 mL/min,about 0.19 mL/min, about 0.2 mL/min, about 0.21 mL/min, about 0.22mL/min, about 0.23 mL/min, about 0.24 mL/min, about 0.25 mL/min, about0.26 mL/min, about 0.27 mL/min, about 0.28 mL/min, about 0.29 mL/min,about 0.3 mL/min, about 0.4 mL/min, about 0.5 mL/min, about 0.6 mL/min,about 0.7 mL/min, about 0.8 mL/min, about 0.9 mL/min, about 1 mL/min,about 2 mL/min, about 3 mL/min, about 4 mL/min, about 5 mL/min, about 6mL/min, about 7 mL/min, about 8 mL/min, about 9 mL/min, or about 10mL/min. In some cases, the flow rate is 0.1 mL/min. In some cases, theflow rate is 0.2 mL/min.

In some aspects, mass spectrometry (e.g., used in 2DLC-MS as describedherein) may refer to electrospray ionization mass spectrometry (ESI-MS).ESI-MS is known in the art as a technique that uses electrical energy toanalyze ions derived from a solution using mass spectrometry. Ionicspecies, including neutral species that are ionized in solution or ingaseous phase, are transferred from a solution to a gaseous phase bydispersal in an aerosol of charged droplets. Subsequently, solventevaporation is conducted to reduce the size of the charged droplets.Then, sample ion is ejected from the charge droplets as the solutionpassing through a small capillary with a voltage relative to ground. Forexample, the wall of the surrounding ESI chamber is performed by mixingthe sample with volatile acid and organic solvent and infusing itthrough a conductive needle charged with high voltage. The chargeddroplets that are sprayed (or ejected) from the needle end are directedinto the mass spectrometer, and are dried up by heat and vacuum as theyfly in. After the drops dry, the remaining charged molecules aredirected by electromagnetic lenses into the mass detector and massanalyzed. In one aspect, the eluted sample is deposited directly fromthe capillary into an electrospray nozzle, for example, the capillaryfunctions as the sample loader. In another aspect, the capillary itselffunctions as both the extraction device and the electrospray nozzle.

In some exemplary embodiments, the electrospray ionization emittercomprises multiple emitter nozzles, such as at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight emitter nozzles, such as two, three, four, five, six, sevenor eight emitter nozzles. In some exemplary embodiments, theelectrospray ionization emitter is a M3 emitter from Newomics (Berkeley,Calif.) which includes 8 emitter nozzles.

In some exemplary embodiments, other ionization modes are used forexample, turbospray ionization mass spectrometry, nanospray ionizationmass spectrometry, thermospray ionization mass spectrometry, sonic sprayionization mass spectrometry, SELDI-MS and MALDI-MS. In general, anadvantage of these methods (like ESI-MS) is that they allow for the“just-in-time” purification of sample and direct introduction into theionizing environment. It is important to note that the variousionization and detection modes introduce their own constraints on thenature of the desorption solution used, and it is important that thedesorption solution be compatible with both. For example, the samplematrix in many applications must have low ionic strength, or residewithin a particular pH range, etc. In ESI, salt in the sample canprevent detection by lowering the ionization or by clogging the nozzle.This problem can be addressed by presenting the analyte in low saltand/or by the use of a volatile salt. In the case of MALDI, the analyteshould be in a solvent compatible with spotting on the target and withthe ionization matrix employed.

In some exemplary embodiments, the electrospray ionization sourceprovides an electrospray with a solvent flow rate of from about 1 μL/minto about 20 μL/min. In various embodiments, the flow rate into the ESIemitter is about 1 μL/min, about 2 μL/min, about 3 μL/min, about 4μL/min, about 5 μL/min, about 6 μL/min, about 7 μL/min, about 8 μL/min,about 9 μL/min, about 10 μL/min, about 11 μL/min, about 12 μL/min, about13 μL/min, about 14 μL/min, about 15 μL/min, about 16 μL/min, about 17μL/min, about 18 μL/min, about 19 μL/min, or about 20 μL/min.

The mass spectrometer can be a native ESI mass spectrometry system. Insome exemplary embodiments, the mass spectrometer can be aquadrupole—Orbitrap hybrid mass spectrometer. The quadrupole-Orbitraphybrid mass spectrometer can be Q Exactive™ Focus HybridQuadrupole-Orbitrap™ Mass Spectrometer, Q Exactive™ Plus HybridQuadrupole-Orbitrap™ Mass Spectrometer, Q Exactive™ BioPharma Platform,Q Exactive™ UHMR Hybrid Quadrupole-Orbitrap™ Mass Spectrometer, QExactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer, Q Exactive™HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer, and Q Exactive™Hybrid Quadrupole-Orbitrap™ Mass Spectrometer. In some exemplaryembodiments, the mass spectrometry system is a Thermo Exactive EMR massspectrometer. The mass spectrometry system can also contain anultraviolet light detector.

A variety of mass analyzers suitable for LC/MS are known in the art,including without limitation time-of-flight (TOF) analyzers, quadrupolemass filters, quadrupole TOF (QTOF), and ion traps (e.g., a Fouriertransform-based mass spectrometer or an Orbitrap). In Orbitrap, abarrel-like outer electrode at ground potential and a spindle-likecentral electrode are used to trap ions in trajectories rotatingelliptically around the central electrode with oscillations along thecentral axis, confined by the balance of centrifugal and electrostaticforces. The use of such instruments employs a Fourier transformoperation to convert a time domain signal (e.g., frequency) fromdetection of image current into a high resolution mass measurement.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1: Characterization of Viral Protein Constituents of AAV Capsids

AAV empty and full samples of different serotypes were preparedin-house. Empty and full AAV samples were mixed and analyzed directlyusing an Agilent 1290 Infinity II 2D-LC system. In the first dimension,the empty and full AAV capsids were separated by a ProPac SAX-10 column(Thermo Scientific). In the second dimension, the AAV capsid was firstdenatured and desalted, and the viral proteins were separated by anACQUITY UPLC Protein BEH C4 column (Waters Corporation). MS analysis ofviral proteins were performed on a Q Exactive™ Plus HybridQuadrupole-Orbitrap mass spectrometer (Thermo Scientific), and the MSdata was analyzed using Xcalibur (Thermo Scientific) and Intact Mass™(Protein Metrics Inc.).

Chemicals and Reagents

Unless otherwise stated, all chemicals and reagents were acquired fromMilliporeSigma (Burlington, Mass., USA). Empty and full capsids of threeAAV serotypes (AAV8, AAV5, and AAV1) were produced in-house at RegeneronPharmaceuticals Inc. (Tarrytown, N.Y., USA), and the detailed sampleinformation and concentrations are shown in Table 1, below. Acetonitrile(ACN) was acquired from Thermo Fisher Scientific (Waltham, Mass., USA).Difluoroacetic acid (DFA) was purchased from Waters Corporations(Milford, Mass., USA). Deionized water (Milli-Q water) was obtained froma Milli-Q integral water purification system (MilliporeSigma).

TABLE 1 Concentration of AAV Samples Sample Concentration AAV8-Empty3.07 × 10¹³ capsids/mL AAV8-GOI1 2.70 × 10¹³ vg/mL AAV8-GOI2 2.70 × 10¹³vg/mL AAV5-Empty 3.32 × 10¹³ capsids/mL AAV5-GOI1 4.61 × 10¹³ vg/mLAAV1-Empty 2.54 × 10¹³ capsids/mL AAV1-GOI1 1.82 × 10¹³ vg/mLvg/mL-viral genome/milliliter

Anion-Exchanqe Chromatography (AEX) Experiment

AAV samples were directly analyzed by AEX without any samplepretreatment. The AEX separation was performed using a Thermo ProPacSAX-10 column (10 μm, 2 mm×250 mm) (Thermo Fisher Scientific) on anACQUITY UPLC I-Class system (Waters Corporation) equipped withfluorescence detector. Mobile phase A (MPA) contained 20 mM bis-trispropane in Milli-Q water, and mobile phase B (MPB) was contained 20 mMbis-tris propane, and 1 M of either tetramethylammonium chloride (TMAC)or tetraethylammonium chloride (TEAC) in Milli-Q water. Both MPA and MPBwere adjusted to pH 9.5 using hydrochloric acid. The flow rate for AEXwas 0.2 mL/min, and the gradient consisted of 10% to 30% MPB from 0 to10 min, 30% to 90% MPB from 10 to 10.1 min, and 90% MPB until 12 min.MPB was reduced to 10% from 12 to 12.1 min, and then maintained at 10%until the end of the 20 min gradient. For all AEX analyses, 1 μL of thesample was injected. Data was recorded using a fluorescence detectorwith excitation (Ex) and emission (Em) wavelengths of 280 nm and 350 nm,respectively.

Reverse-Phase Liquid Chromatography (RPLC) Experiment

AAV samples were denatured with 10% acetic acid for 10 minutes prior toRPLC analysis. The RPLC experiment was performed using an ACQUITY UPLCProtein BEH C4 column (1.7 Em, 300 Å, 2.1 mm×150 mm) (WatersCorporation) on an ACQUITY UPLC I-Class system (Waters Corporation)equipped with a fluorescence detector. MPA was prepared with 0.1% DFA inMilli-Q water and MPB was prepared with 0.1% DFA in acetonitrile. Thegradient was run at 0.2 mL/min starting with 20% to 32% MPB from 0 to 1min, followed by 32% to 36% MPB from 1 to 16 min, 36% to 80% MPB from 20to 21.5 min, 80% to 20% MPB from 21.5 to 22 min, and then 20% MPB untilthe end of the 30 min gradient. For all RPLC analyses, 1 μL of thesample was injected. Data was acquired using a fluorescence detectorwith 280 nm Ex wavelength and 350 nm Em wavelength.

Two-Dimensional Liquid Chromatography (2DLC) Conditions

The 2DLC experiment was performed on an Agilent 1290 Infinity II 2D-LCSystem (Agilent Technologies, Santa Clara, Calif., USA). The AEXgradient was applied to the first dimension at a flow rate of 0.1 mL/mininstead of 0.2 mL/min.. At this lower flow rate, the 40 μL trapping loopallowed for 0.4 seconds of sample trapping. 6 μL of the AAV8 samplecontaining the gene of interest 1 (GOI1) was injected. Heart-cutting wasperformed using the time-based mode for high-resolution sampling, wherepeaks were selected based on the UV spectrum at 280 nm wavelength. Thesample was then transferred to the second-dimension RP column from thetrapping loop. The RPLC gradient was also applied to the seconddimension, with the addition of 10-minute holding period at 80% MPA toremove the MS-incompatible salt used fpr AEX separation and onlinedenaturation of intact viral capsids. An ACQUITY UPLC Protein BEH C4column (1.7 μm, 300 Å, 2.1 mm×50 mm) (Waters Corporation) was used as atrap column prior to the analytical column with 150 mm in length. Adivert valve was utilized to direct flow to waste during denaturationand salt removal and to the analytical column for downstream analysis.To maintain the temperature of the analytical column, an additional LCpump was used to keep the starting RPLC mobile phase at 0.05 mL/min.

Mass spectrometry (MS) Data Acquisition

RPLC-MS data was acquired using a Thermo Scientific Q Exactive™ PlusHybrid Quadrupole-Orbitrap mass spectrometer (Bremen, Germany). For dataacquisition, the resolution was set at 17,500, AGC target at 3e6, andmaximum injection time at 500 ms. The spray voltage was set at 3.8 kVand S-lens RF level was at 50. The sheath and auxiliary gas flow were 40and 15, respectively, and the capillary temperature and auxiliary gasheater temperatures were both set at 250° C. Spectra were acquired from1,000 to 3,000 m/z.

All 2DLC-MS data were acquired on a Thermo Scientific Orbitrap Exploris480 mass spectrometer (Bremen, Germany) equipped with a ThermoScientific NanoSpray Flex ion source. A nano flow splitter fromAnalytical Scientific Instruments (Richmond, CA, USA) was set at 50, andelectrospray ionization emitter tips (CoAnn Technologies, Richland, WA,USA) were used for electrospray. For data acquisition, the resolution,AGC target, maximum injection time, and number of microscans were set at15,000, 3e6, auto, and 3, respectively. The spray voltage was set to2,200 V, RF lens level was at 50%, and the ion transfer tube temperaturewas set to 275° C. Mass spectra were acquired from 1,000 to 3,000 m/z.

Data Analysis

For data acquired on a Waters instrument, the analysis was performed onthe Empower 3 version 1.65. Mass spectrometry data were analyzed usingXcalibur 4.3.73.11. Data acquired on the Agilent instrument wereanalyzed using OpenLAB CDS ChemStation Edition Rev. C.01.07 SR2. Intactmass analysis was performed using Intact Mass™ version 3.11-1 (ProteinMetrics Inc., Cupertino, Calif., USA).

Results and Discussion

A 2DLC-MS platform (schematic illustrated in FIG. 2B) was utilized forAAV characterization. The method implemented high-resolution AEX in thefirst dimension for empty and full virus capsid separation (FIGS. 3A, 3Band 5 ). Following online denaturation and desalting of MS-incompatiblesalt, the viral proteins were subjected to intact protein separation inthe second RPLC dimension and intact protein characterization by MS(FIGS. 3C, 3D, 6, 7A, 7B, 7C, 7D, 8A and 8B).

In the first dimension, the separation of empty and full AAV capsids wasperformed using AEX. With the salt gradient using eithertetramethylammonium chloride and tetraethylammonium chloride compared totraditionally used sodium chloride, the empty and full AAV capsids werebaseline resolved for all the tested samples. Empty capsids, and capsidscontaining a gene of interest (GOI) were separated for each of AAV1,AAVS and AAV8 serotypes (FIGS. 3A and 3B). The separation of empty andfull capsids allowed for quantitation of the relative percentage ofempty and full capsids in the samples (e.g., FIG. 5 ), which wereconsistent with those determined by AUC. Following high-resolutionseparation of empty and full capsids in the first dimension, onlinetrapping was performed to select the peak of interest. Intact AAV capsidwas denatured into individual viral proteins prior to the RPLC analysisin the second dimension. A starting mobile phase composition of the RPLCwas used to both denature the AAV capsids through acidification andremove MS-incompatible salt used in the AEX separation. In the seconddimension, the viral proteins were separated by RPLC usingdifluoroacetic acid as the ion-pairing reagent. MS analysis of the viralproteins revealed low-abundant species including unmodified,phosphorylated, and oxidative proteoforms. Additionally, differences inthe phosphorylation levels of VP2 were observed among AAV samples (see,e.g., FIGS. 7A, 7B, 7C, 7D, 8A and 8B).

The AAV capsid comprises three types of viral protein (VP) subunits,VP1, VP2 and VP3, totaling 60 copies in a ratio of 1:1:10 (VP1:VP2:VP3).These capsid proteins are alternatively spliced from one mRNA, and thusshare a common sequence.

In the reverse-phase liquid chromatography-mass spectrometry (RPLC-MS)analysis, the major proteoforms included acetylated VP1 and itsphosphorylated form, VP2 and its phosphorylated form, acetylated VP3,and VP3 clip species. Minor proteoforms included those arising fromcleavage of an aspartic acid-proline (DP) bond. This DP bond cleavagegenerates species including acetylated VP1 clip, VP2 clip and itsphosphorylated form, and acetylated VP3 clip, and a DP clip fragment.The clip species arise from cleavage of an aspartic acid-proline (DP)bond, which may be introduced during denaturation and separation.Additionally, unmodified and oxidized VP3 species were observed, alongwith additional acetylated VP3 clip species in which an asparticacid-glycine (DG) bond was broken (FIG. 8A).

Similarly, RPLC-MS analysis for an AAV1-Empty capsid sample revealed themajor proteoforms included acetylated VP1 and its phosphorylated form,VP2 and its phosphorylated form, acetylated VP3, and VP clip arisingfrom DP bond cleavage. DP bond cleavage also generated proteoforms suchas VP2 clip and its phosphorylated form, and acetylated VP3 clip, andclip fragment. Low-abundant oxidized proteoforms were detected for allthree VPs. DG bond cleavage was also observed, providing an additionalacetylated VP3 clip species and a DG clip fragment (FIG. 8B).

In addition to proteoform identification, intact mass analysis alsorevealed differences in post-translational modification (PTM) level.Previous studies have shown Ser149 to be the major phosphorylation sitein the AAV8 sequence. As the three viral proteins are alternativelycleaved, Ser149 was not included in the VP3 sequence. While thephosphorylation level in VP1 remained similar among three AAV8 samples,the phosphorylation level in VP2 varied significantly (FIG. 8A). BothAAV8 samples containing GOI showed elevated VP2 phosphorylation levelscompared to the AAV8 sample without GOI. For AAV1, phosphorylationdifferences were not observed in viral proteins for the empty and fullcapsids (FIG. 8B).

The 2DLC-MS method demonstrated herein enabled high throughput andmulti-attribute AAV characterization in a single system. In the firstdimension, AEX provided high resolution separation of empty and fullcapsids using TMAC or TEAC. Online denaturation and desalting wereachieved to dissociate the AAV capsids into viral proteins. In thesecond dimension, RPLC coupling to MS was used to characterize the viralproteins. Using this method, AAV samples were directly analyzed withoutsample pretreatment to minimize sample handling and avoid sample loss.The platform combined two characterization techniques in one analysisand provided good separation and high sensitivity, enabling detection ofboth major and minor viral protein proteoforms and fragments.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A method for identifying viral protein constituents of a sample ofviral particles, comprising: (a) subjecting the sample of viralparticles to first-dimension chromatography to separate intact viralcapsid components of the sample; (b) subjecting at least a portion ofthe intact viral capsid components to online denaturation to yieldindividual intact viral proteins; (c) subjecting the intact viralproteins to second-dimension chromatography to separate the intact viralproteins; and (d) determining the masses of the separated intact viralproteins to identify the viral protein constituents of the sample ofviral particles.
 2. The method of claim 1, further comprising selectinga portion of the separated intact viral capsid components, whereinsubjecting at least a portion of the intact viral capsid components toonline denaturation to yield individual viral proteins comprisessubjecting the selected portion of the separated intact viral capsidcomponents to online denaturation.
 3. The method of claim 1, whereinsample of viral particles comprises adeno-associated virus (AAV)particles.
 4. The method of claim 3, wherein the AAV particles are ofserotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ,AAV-DJ/8, AAV-Rh10, AAV-retro, AAV-PHP.B, AAV8-PHP.eB, or AAV-PHP.S. 5.(canceled)
 6. The method of claim 1, wherein the intact viral capsidcomponents comprise empty viral capsids and full viral capsids.
 7. Themethod of claim 1, wherein the first-dimension chromatography comprisesion-exchange chromatography or anion-exchange chromatography, and thesecond-dimension chromatography comprises reverse-phase chromatographyor hydrophilic interaction liquid chromatography. 8-11. (canceled) 12.The method of claim 3, wherein the viral protein constituents comprise(a) VP1, VP2 and/or VP3 of an AAV particle, or (b) post-translationalvariants of VP1, VP2 and/or VP3.
 13. (canceled)
 14. The method of claim12, wherein the post-translational variants of VP1, VP2 and/or VP3comprise (a) acetylated, phosphorylated and/or oxidized variants of VP1,VP2 and/or VP3, or (b) fragments of VP1, VP2 and/or VP3 produced fromcleavage of an aspartic acid-proline bond and/or cleavage of an asparticacid-glycine bond.
 15. (canceled)
 16. The method of claim 1, furthercomprising: (a) detecting the intact viral capsid components separatedby the first dimension chromatography, and identifying a ratio of emptyviral capsids to full and partially-full viral capsids; and/or (b)detecting the intact viral proteins separated by the second-dimensionchromatography, and quantifying the relative abundance of the viralprotein constituents of the sample of viral particles.
 17. (canceled)18. The method of claim 16, wherein the intact viral capsid componentsand/or the intact viral proteins are detected using an ultraviolet orfluorescence detector.
 19. A method for identifying viral proteinconstituents of a sample of adeno-associated virus (AAV) particles,comprising: (a) subjecting the sample of AAV particles to anion-exchangechromatography to separate intact viral capsid components in the sample,wherein the intact viral capsid components comprise intact empty viralcapsids and intact full viral capsids comprising a heterologous nucleicacid molecule; (b) selecting a portion of the intact viral capsidcomponents for online desalting and denaturation; (c) subjecting theselected portion of the intact viral capsid components to onlinedesalting and denaturation to yield individual intact viral proteins,wherein the intact individual viral proteins comprise VP1, VP2, VP3 andat least one variant of VP1, VP2 or VP3; (d) subjecting the intact viralproteins to reverse-phase liquid chromatography or hydrophilicinteraction liquid chromatography to separate the intact viral proteins;and (e) determining the masses of the separated intact viral proteins toidentify the viral protein constituents of the sample of AAV particles.20. The method of claim 19, further comprising: (a) detecting the intactviral capsid components separated by the anion-exchange chromatography,and identifying a ratio of empty viral capsids to full andpartially-full viral capsids; and/or (b) detecting the intact viralproteins separated by the reverse-phase liquid chromatography orhydrophilic interaction liquid chromatography, and quantifying therelative abundance of the viral protein constituents of the sample ofAAV particles. 21-22. (canceled)
 23. The method of claim 19, wherein theAAV particles are of serotype AAV1 or AAV8.
 24. The method of claim 19,wherein the at least one variant of VP1, VP2 or VP3 comprises apost-translational variant of VP1, VP2 or VP3.
 25. The method of claim24, wherein the post-translational variant of VP1, VP2 or VP3 comprises:(a) an acetylated variant of VP1, VP2 or VP3; (b) a phosphorylatedvariant of VP1, VP2 or VP3; (c) an oxidized variant of VP1, VP2 or VP;(d) a fragment of VP1, VP2 or VP3 produced from cleavage of an asparticacid-proline bond; and/or (e) a fragment of VP1, VP2 or VP3 producedfrom cleavage of an aspartic acid-glycine bond. 26-29. (canceled) 30.The method of claim 20, wherein the intact viral capsid componentsand/or the intact viral proteins are detected using an ultraviolet orfluorescence detector.
 31. The method of claim 19, wherein determiningthe masses of the separated intact viral proteins comprises subjectingthe separated intact viral proteins to electrospray ionization massspectrometry.
 32. The method of claim 19, wherein the intact viralproteins are subjected to reverse-phase liquid chromatography orhydrophilic interaction liquid chromatography.
 33. (canceled)
 34. Themethod of claim 19, wherein the intact viral capsid components of thesample subjected to anion-exchange chromatography are separated: (a)using a first mobile phase comprising from 15 mM to 25 mMbis-tris-propane (BTP), from 250 mM to 1 M tetramethylammonium chloride(TMAC), and from 1 mM to 3 mM magnesium chloride at a pH of from 8 to 9;(b) using a first mobile phase comprising 20 mM ±2 mM BTP, 500 mM ±50 mMTMAC, and 2 mM ±0.2 mM MgCl₂ at a pH of 8.5±0.1; (c) using a firstmobile phase comprising from 15 mM to 25 mM bis-tris-propane (BTP), from250 mM to 1 M tetramethylammonium chloride (TMAC), and from 1 mM to 3 mMmagnesium chloride at a pH of from 8 to 9 or a first mobile phasecomprising 20 mM ±2 mM BTP, 500 mM ±50 mM TMAC, and 2 mM ±0.2 mM MgCl₂at a pH of 8.5±0.1, and a second mobile phase comprising from 15 mM to25 mM bis-tris-propane (BTP), and from 1 mM to 3 mM magnesium chlorideat a pH of from 8 to 9; (d) using a first mobile phase comprising from15 mM to 25 mM bis-tris-propane (BTP), from 250 mM to 1 Mtetramethylammonium chloride (TMAC), and from 1 mM to 3 mM magnesiumchloride at a pH of from 8 to 9 or a first mobile phase comprising 20 mM±2 mM BTP, 500 mM ±50 mM TMAC, and 2 mM ±0.2 mM MgCl₂ at a pH of8.5±0.1, and a second mobile phase comprising 20 mM ±2 mM BTP, and 2 mM±0.2 mM MgCl₂ at a pH of 8.5±0.1; or (e) using a first mobile phasecomprising from 15 mM to 25 mM bis-tris-propane (BTP), from 250 mM to 1M tetramethylammonium chloride (TMAC), and from 1 mM to 3 mM magnesiumchloride at a pH of from 8 to 9 or a first mobile phase comprising 20 mM±2 mM BTP, 500 mM ±50 mM TMAC, and 2 mM ±0.2 mM MgCl₂ at a pH of8.5±0.1, and a second mobile phase comprising from 15 mM to 25 mMbis-tris-propane (BTP), and from 1 mM to 3 mM magnesium chloride at a pHof from 8 to 9 or a second mobile phase comprising 20 mM ±2 mM BTP, and2 mM ±0.2 mM MgCl₂ at a pH of 8.5±0.1, and a third mobile phasecomprising from 1.5 M to 2.5 M sodium chloride or a third mobile phasecomprising 2 M ±0.1 M sodium chloride. 35-39. (canceled)
 40. The methodof claim 34, wherein the separation of the intact viral capsidcomponents is (a) performed with a mobile phase gradient, or (b)performed with a mobile phase gradient comprising, in sequence: 10%first mobile phase and 90% second mobile phase for 1 minute; increasingthe first mobile phase from 10% to 42%, and decreasing the second mobilephase from 90% to 58%, over a period of 20 minutes; 100% third mobilephase for 5 minutes; and 10% first mobile phase and 90% second mobilephase for 10 minutes. 41-42. (canceled)